Conversion of glucose to vitamin C (ascorbic acid) is a complicated process because it involves the selective epimerization, oxidation, and lactone formation. The natural biosynthetic pathways are long and incorporate many energy-consuming reactions (Davey, et al., Plant Physiol. 121 (2):535-43 (1999); Nishikimi, M and K. Yagi, Subcell Biochem. 25:17-39 (1996); Wheeler, et al., Nature 393(6683):365-9 (1998). The current commercial process for ascorbic acid production (the Reichstein process) couples a single, initial biological step—the microbial reduction of glucose to sorbitol—with subsequent, multi-step chemical conversion of blocked derivatives of sorbitol to ascorbic acid (Crawford, T. C., American Chemical Society, Washington, D.C. (1982); Reichstein, T. and A. Grussner, Helv. Chim. Acta 16:311 (1934)). An alternative commercial process has been proposed that consists of biological conversion of glucose to 2-keto-L-gulonic acid which is lactonized chemically to ascorbic acid (Anderson, et al., Science 230:144-149 (1985); Grindley, et al., Appl. Environ. Microbiol. 54:1770-1775 (1988); Sonoyama, et al., U.S. Pat. No. 3,922,194 (1975)). The biological metabolism involved is simpler than that of natural biosynthetic routes and requires less metabolic energy (less ATP and NADPH). In this process, glucose is first converted to 2,5-diketo-D-gluconic acid by endogenous oxidases of a suitable bacterial strain using molecular oxygen as the ultimate electron acceptor. 2,5-diketo-D-gluconic acid is then reduced enzymatically to 2-keto-L-gulonic acid by a heterologous 2,5-diketo-D-gluconic acid reductase (DKGR) expressed in the production strain. The NADPH required for the reaction is generated by the metabolism of the host strain. Finally, chemical lactonization of 2-keto-L-gulonic acid generates ascorbic acid.
To date, only two 2,5-diketo-D-gluconic acid reductases have been extensively characterized, both isolated from a species of Corynebacterium (Miller, et al., J. Biol. Chem. 262(19):9016-20; Powers, D. B. and S. Anderson, U.S. Pat. No. 5,795,761 (1998); Sonoyama, T. and K. Kobayashi, J. Ferment. Technol. 65:311-317 (1987)). These enzymes are able to reduce 2,5-diketo-D-gluconic acid, but alternative or altered reductases could improve ascorbic acid production by the process described above or variations of it. Both of the Corynebacterium enzymes are relatively inefficient catalysts, exhibiting Km values for 2,5-diketo-D-gluconic acid greater than 1 mM and catalytic efficiencies (kcat/Km) less than 20 mM−1 sec−1.
2,5-diketo-D-gluconic acid reductases are members of the aldo-keto reductase superfamily (Jez, et al., Biochem J. 326 (Pt3):625-36 (1997); Seery, et al., J Mol. Evol. 46(2):139-46 (1998)). Like almost all other aldo-keto reductases, the known 2,5-diketo-D-gluconic acid reductases are exclusively specific for NADPH (Jez, et al., Biochem J. 326 (Pt3):625-36 (1997); Seery, J Mol Evol. 46(2):139-46 (1998)). Recently, additional aldo-keto reductases that can convert 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid have been isolated from E. coli based on a search of the genome sequence (Yum, et al., Bacteriol. 180 (22):5984-8 (1998); Yum, et al., Appl Environ Microbiol. 65 (8):3341-6 (1999)). However, these enzymes also catalyze the reaction relatively inefficiently. The known 2,5-diketo-D-gluconic acid reductases also lack stability; both Corynebacterium enzymes are thermally labile (Powers, D. B. and S. Anderson, U.S. Pat. No. 5,795,761 (1998); Sonoyama, T. and K. Kobayashi, J. Ferment, Technol. 65:311-317 (1987)).
It would therefore be desirable to solve the problem of inefficient reductases by providing 2,5-diketo-D-gluconic acid reductases which are more efficient than known reductases. In particular, it would be desirable to provide novel enzymes which display greater catalytic efficiency than previously known 2,5-diketo-D-gluconic acid reductases, and which have NADH-dependant activity. It would further be desirable for the reductase to be more stable thermally than known 2,5-diketo-D-gluconic acid reductases. It would further be desirable to provide variants of said reductases, methods of making, screening and using novel reductases.